Assessment of laboratory methods for quantifying aqueous bacterial diffusion

Abstract

Laboratory studies of bacterial transport and attenuation in a porous media were conducted under low velocity and near diffusion-dominated conditions. This thesis reviews and summarizes velocity data from a great deal of pertinent bacterial transport and attenuation investigations. Peclet numbers calculated from the literature data and data from this study correlated well with the Peclet plot. Three independent methods were investigated for determining bacterial diffusion rates: dynamic light scattering measurements, the diffusing sphere technique, and the double reservoir diffusion technique. One motile and four non-motile bacteria were used in these mono-culture experiments: Pseudomonas fluorescens 840406-E, Pseudomonas fluorescens m18, Klebsiella oxytoca, Burkholderia cepacia G4PR1, and a Pseudomonas isolate. Dynamic light scattering measurements were used to measure the Brownian movement of the bacteria. These measurements yielded free-water bacterial diffusion coefficients of ~1x10-13 m2·sec-1 for all five bacteria. Estimates of bacterial diffusion coefficients using the Einstein-Stokes and the volume-fraction modified Einstein-Stokes equation agreed with measurements made using dynamic light scattering. The diffusing sphere model and technique was re-designed to measure free-water bacterial diffusion. The diffusion coefficient for fluorescein (4.4x10-10 m2·sec-1), found by simulating diffusing fluorescein results, corresponded well with published values for the free-water diffusion of fluorescein. Many attempts were made to adapt this technique to measure free-water bacterial diffusion coefficients. Rapid sinking of the diffusing sphere prevented measurement of the bacterial diffusion and the technique could not be used to measure free-water bacterial diffusion. The double reservoir diffusion cell model was designed and constructed to study the diffusion-dominated transport and attenuation of bacteria through saturated quartz sand. While the double reservoir diffusion technique was successful for determining an effective diffusion coefficient of chloride (6.1x10-10 m2·sec-1), the technique could not be adapted for measurement of bacterial diffusion in this study. The inability of the double reservoir diffusion test to quantify bacterial diffusion through saturated quartz sand was attributed to the difficulty in the enumeration of bacterial concentrations without disturbing experimental conditions. As well, the low values for bacterial diffusion (~1x10-13 m2·sec-1) provided a bacterial diffusive flux low enough that experimental errors may have influenced the results. Achieving and maintaining adequate experimental conditions for the laboratory study of diffusion-dominated bacterial transport and attenuation remains one of the most daunting challenges facing researchers in this field today.